Conventionally, various automatic cell culture apparatuses have been supplied. For example, Patent Document 1 pertaining to a prior application of the present applicants discloses an automatic cell culture system. The automatic cell culture system can handle a plurality of culture cassettes containing a culture dish, automatically transfer the culture cassette with a robot arm for a dispensing operation and the like, control atmospheres such as gas concentration, temperature, and humidity in an incubator, and reliably prevent not only contamination in the incubator but also contamination during transfer of the culture dish for a dispensing operation and the like.
Patent Documents 2 and 3 also disclose an automatic cell culture apparatus that automates, for example, an exchange operation of a liquid culture medium with a general purpose multiple joints type robot in a two-dimensional culture apparatus. The automatic cell culture apparatus has an effect on the prevention of bacterial invasion by minimization of human intervention, but an expensive general purpose industrial robot performs the same operations as those that a human has performed, and such an apparatus requires high cost. Moreover, such an apparatus is not beyond common two-dimensional cell culture apparatuses and is not an apparatus for achieving three-dimensional culture.
Meanwhile, mesenchymal stem cells derived from the bone marrow has multipotency to be differentiated into tissues such as bone, cartilage, adipose, and ligament. However, it is known that, in a common in vitro culture, the cells are settled to the bottom of a petri dish due to the Earth's gravity to form a two-dimensional sheet and the original cell characteristics disappear. To address this, a culture method using a rotating wall vessel (RWV) bioreactor has been developed in order to achieve the three-dimensional culture in a microgravity environment that is nearly gravity-free, and cells are three-dimensionally cultured with appropriate differentiation inducing factors to be differentiated into an original tissue of the cell. A culture apparatus using the RWV is released from Synthecon. The apparatus is composed of a flat cylindrical vessel having a gas permeable membrane on the back side, and the vessel is rotated around an attaching part to which a horizontal rotating shaft of a rotation controller is attached. The rotation of the vessel constantly changes the gravity direction to cells, and consequently can generate a microgravity environment having one-hundredth the ground gravity by time average. Hence, clumps of cells are not settled and can be cultured while floating. The apparatus is typically referred to as a rotating culture device.
The RWV used in the conventional rotating culture device includes an inlet/outlet for a cell suspension and a pair of a supply port and a discharge port for a liquid culture medium on the front face orthogonal to the rotating shaft, and the liquid culture medium is exchanged manually. That is, for liquid culture medium exchange, the RWV is removed from the rotating shaft and left with the supply port and the discharge port facing upward, a rubber cap of the supply port is taken off, a leading end of a supply syringe including a new liquid culture medium is closely connected to the supply port, while a leading end of an empty discharge syringe is closely connected to the discharge port, then cocks of the supply port and the discharge port are unlocked, a piston of the supply syringe is pushed by one hand to supply the liquid culture medium into the vessel, and simultaneously a piston of the discharge syringe is pulled by the other hand to suck the used liquid culture medium in the vessel. The culture period of cells is typically about two weeks, and a culture medium is required to be exchanged every several days. Thus, the culture medium exchange operation that is complicated and requires a lot of skill must be carried out in each case. Such a manual culture medium exchange operation requires a lot of labor and time. When many RWVs are run, the exchange operation becomes a heavy burden as well as unexpected environmental contamination may occur during the culture medium exchange operation. In addition, the vessel having the cell suspension inlet, the supply port, and the discharge port on the front face has an disadvantage of small view for the observation of cells in the vessel.
To address this, the present applicant has developed an automatic cell culture apparatus as disclosed in Patent Document 4. Namely, the automatic cell culture apparatus includes a closed housing and a rotating culture device in the closed housing. The rotating culture device includes a cylindrical culture container having a horizontal rotating shaft, a cell suspension inlet, a supply port, and a discharge port, and each of the supply port and the discharge port has a septum seal structure for liquid culture medium exchange. The supply port and the discharge port are provided as a pair on the outer circumferential cylindrical face of the culture container at positions 180° apart from each other with respect to the rotating center toward the radial direction. The automatic cell culture apparatus also includes a syringe shifting means for inserting/withdrawing an injection needle of a supply syringe containing a new liquid culture medium to/from the supply port and for inserting/withdrawing an injection needle of an empty discharge syringe to/from the discharge port while locating the supply port and the discharge port on a vertical line with the supply port facing upward and includes a piston driving means for pushing a piston of the supply syringe while inserting the supply syringe into the supply port and for simultaneously pulling a piston of the discharge syringe while inserting the discharge syringe into the discharge port.
The apparatus thereby has a function of automatically exchanging a liquid culture medium in the rotating culture device using the RWV. Hence, such an apparatus can largely reduce burdens of culture operators such as researchers and achieve efficient cell culture as well as minimize the possibility of contamination in culture. When a plurality pairs of the supply ports and the discharge ports are provided on the outer circumferential cylindrical face of the culture container at an equal angle interval depending on a culture period and the number of liquid culture medium exchanges, the supply port and the discharge port can be newly used for every liquid culture medium exchange. Thus, such an apparatus has an advantage of minimizing the contamination during the liquid culture medium exchange.